Project Summary Dopamine (DA) is a powerful neuromodulator that facilitates memory formation and underlies reward-related behaviors by regulating synaptic plasticity. Microdialysis and voltammetry are commonly used to measure extracellular concentrations of neuromodulators in vivo, but these techniques suffer from poor temporal resolution and neurotransmitter specificity, respectively. Quantifying DA release alone provides limited information as DA can exert various effects on synaptic plasticity depending on the timing of release, concentration of DA released, and the receptor subtypes that are activated. Most studies define the DA receptor subtypes that mediate plasticity by pharmacological isolation, but this approach suffers from lack of specificity, widely perturbs DA signaling, and can produce off target effects. Therefore, a tool to assess DA signaling in vivo with high spatiotemporal resolution and receptor subtype specificity will provide great insight into the cellular mechanisms by which DA modulates plasticity with cell-type specificity in behaving animals. I propose to create biosensors to visualize the dynamic activity of distinct DA receptors in cells, brain slices, and in vivo with high spatiotemporal resolution using 2-photon fluorescence lifetime imaging microscopy (2pFLIM). In Aims 1 and 2, I will engineer FRET-based sensors for each DA receptor subtype by screening and validating constructs first in cell lines and then in brain slices. In Aim 3, I will employ these sensors to determine how activity patterns of DA receptor subtypes influence long-term structural plasticity of dendritic spines in cortex during motor learning. We predict that activation of different DA receptor subtypes will exert distinct effects on the stability of newly formed spines following learning.